Project Summary. Bacteriophages play a major role in bacterial evolution, in mediating bacterial pathogenicity and antibiotic resistance, in modulating the human microbiome and they have great potential as nanotherapeutics. Understanding these issues with respect to human disease and harnessing their potential as theranostic agents requires a fundamental understanding of virus development. The genome packaging pathways are strongly conserved in the large double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic. In this broad class of viruses, a terminase enzyme is responsible for (i) excision of an individual genome from concatemeric substrate (genome maturation) and (ii) translocation of DNA into a procapsid shell (genome packaging). These functions are catalyzed by terminase enzymes assembled into discrete maturation and packaging motor complexes. Terminases are composed of a catalytic subunit and a DNA recognition subunit, both of which are essential for genome packaging in vivo. Structural and single-molecule studies have provided insight into packaging motor complexes composed of the catalytic subunit in isolation; however, there is little information on motor complexes containing both essential subunits. Further, there is a dearth of structural information on the equally essential maturation complex precursor. This is due, in part, to the absence of well-characterized holoenzyme preparations and a dearth of in vitro assays to comprehensively assess the pathway. We have developed rigorous assays in which the biochemical, biophysical and structural features of the lambda genome-packaging pathway can be defined in great detail. Using these tools, we propose to characterize the structural (cryo-electron microscopy) and functional (biophysical, kinetic) features of the maturation complex, which show mechanistic similarity to the tetrameric type IIE/F restriction endonucleases. We directly address an emerging controversy relating to the DNA architecture in the maturation complex that mediates complex stability. We next test the hypothesis that the lambda motor also functions as a tetrameric complex and that ATP hydrolysis by the motor is strongly cooperative; these features represent a significant departure from currently accepted paradigms. Finally, we characterize a putative nucleotide switch mechanism that controls the transition from the stable maturation complex to the dynamic motor complex bound to the capsid and we rigorously define the energy budget of the translocating motor. The proposed studies will provide structural and mechanistic detail on two sequential packaging complexes and their transition through the genome-packaging pathway. These features are shared by all of the dsDNA viruses that package genomes from concatemeric precursors (phage, herpes) and the results will be of broad and general significance.